In North America, the incidence of Protozoan infection is 2–3% overall, 30–50% in children's day care situations. Outside of North America, incidence of Protozoan infection has been reported as high as 40–60% in developing countries. Some waterborne outbreaks occur without the availability of effective drugs for treatment.
Protozoan infection is usually treated with drugs, which are costly and may have dangerous side effects in certain individuals. Individuals with immunodeficiency diseases, such as HIV and some cancers, have a high susceptibility to protozoan infection and these individuals cannot tolerate the toxic side effects of drugs currently used to treat protozoan infection.
Transmission of a pathogenic microorganism from an animals to a human, termed zoonosis, is responsible for significant outbreaks of infectious disease in human populations. An outbreak of Cryptosporidium in Milwaukee (Wis.), during which more than 400,000 people became infected, was believed to be caused by contamination of the water reservoir by cattle feces containing viable Cryptosporidium parvum oocytes. A similar outbreak occurred in North Battleford (Canada) in 2002. The water borne transmission of E. coli, an intestinal bacterium, caused significant morbidity and mortality of humans in Walkerton (Canada). Outbreaks of “beaver fever” caused by the protozoan parasite, Giardia lamblia, have occurred in Banff and Edmonton (Canada) in the recent past. Host nutritional and immune status is closely related with the disease course of giardiasis.
Giardia is a protozoan parasite that inhabits the upper small intestine of a wide range of vertebrates including humans. It is spread via contaminated food and water and by direct host to host contact. After entering the host, the parasites emerge from the cysts, and adhere to the epithelial brush border of the small intestine as flagellated trophozoites. The trophozoites multiply in the small intestine, eventually encysts and are passed in the feces as infectious cysts. The number of cysts released in feces was reported to be related to the trophozoite burden in the small intestine and degree of pathology observed during the infection (Belosevic and Faubert, 1983). A full citation for each prior art document referenced herein is provided below.
Clinical manifestations of giardiasis range from asymptomatic to symptomatic. Symptoms include diarrhea, weight loss, abdominal distension, vomiting and abdominal pain (Farthing 1996; Wolfe 1992). The severity of symptoms may vary and was found to be related to the initial number of cysts ingested, the age of the host, and the state of the host immune system. Disaccharidase deficiency causing malabsorption has been observed in both humans (Jennnings 1976) and animals (Buret et al., 1991; Daniels and Belosevic, 1995; Gillon et al., 1982), and was related to the parasite burden in the small intestine (Daniels and Belosevic, 1995).
Gangliosides, sialic acid-containing glycosphingolipids, are located at the surface of the cell membrane with the hydrophilic oligosaccharide chain extending into the extracellular space. Glycosphingolipid constitutes approximately 20% of the brush border membrane lipids (Forstner and Wherrett, 1973). The dominant ganglioside is GM3 (Daniels and Belosevic, 1995) which is 7 times more concentrated in the neonatal compared to adult intestine of rats (Bouhours and Bouhours, 1983). The specific physiological roles of gangliosides are poorly understood, however, studies showed that gangliosides provide binding sites for a wide range of pathogens including viruses, bacteria and fungi (Holmgren et al., 1985; Kyogashima et al., 1989; Laegreid and Otnaess, 1987; and Rolsma et al., 1998). For example, ganglioside GM3 acts as a natural receptor in pig small intestine for rotavirus (Rolsma et al., 1998) and the enterotoxigenic bacteria Escherichia coli (E. coli) K99 (Kyogashima et al., 1989). Ganglioside GM1 in human intestine (Holmgren et al., 1985) and in human milk (Laegreid and Otnaess, 1987) also provides receptors for enterotoxin of Vibrio cholerae and the heat-labile E. coli, thereby acting as a physiological barrier for protection against these enteric infections.
Preterm newborn infants fed ganglioside supplemented formula at a concentration of 1.43 mg/100 Kcal, were shown to have significantly lower numbers of E. coli and bifidobacteria in the feces (Rueda et al., 1998). Previous studies showed that gangliosides exist in clusters in the plasma membrane forming glycosphingolipid enriched domains (Buret et al., 1991), and that these domains are the preferential interaction sites between target cells and pathogens (Karlsson, 1995).
Decreased prevalence of giardiasis among infants fed breast milk containing high titers of anti-Giardia secretory IgA (sIgA) has been reported (Walterspiel et al., 1994). Studies showed that non-immune components of human milk such as conjugated bile salts (Gillin, 1987), unsaturated fatty acids (Rohrer et al., 1986) and free fatty acids (Reiner et al., 1986) may be involved in the elimination of the parasites. Although breast milk also contains a significant amount of gangliosides (Rueda et al., 1996), it has never been examined whether gangliosides may play a protective role in giardiasis.
It is desirable to find a compound, a class of compounds, or composition active against giardiasis.
Prior art references referred to herein are provided below:                Belosevic and Faubert. 1983. Exp. Parasitol. 56:93–100.        Bouhours and Bouhours. 1983. J. Biol. Chem. 258:299–304.        Brown and Rose. 1992. Cell. 68:533–544.        Buret et al., 1991. Parasitol Res. 77:109–114.        Clandinin and Yamashiro. 1982. J. Nutr. 112: 825–828.        Daniels and Belosevic. 1995. Parasitol Res. 81:143–147.        Diamond et al., 1978. Trans. R. Soc. Trop. Med. Hyg. 72:431–432.        Farthing. 1996. Giardiasis. Gastro. Clin. North Am. 25:493–515.        Folch and Sloane-Stanley. 1957. J. Biol. Chem. 226:497–509.        Forstner and Wherrett. 1973. Biochim. Biophys. Acta. 306:446–459.        Gibson et al., 1999. Exp. Parasitol. 92:1–11.        Gillin. 1987. Exp. Parasitol. 63:74–83        Gillin et al., 1985. Infect. Immun. 47:619–622.        Gillon et al., 1982. Gut. 23:498–506.        Holmgren et al., 1985. Gasteroenterology. 89:27–35.        Iwamori et al., 1984. J. Biochem. 95:761–770.        Jarrol et al., 1981. Mol. Biochem. Parasitol. 2:187–196.        Jennnings et al., 1976. Aust. NZ J. Med. 6:556–560.        Karlsson 1995. Curr. Opin. Structur. Bio.5:622–635.        Kyogashima et al., 1989. Arch. Biochem. Biophys. 270:391–397.        Laegreid and Otnaess. 1987. Life Sci. 40:55–62.        Ortega-Barria et al., 1994. J. Exp. Med. 94:2283–2288.        Reiner et al., 1986. J. Infect. Dis. 154:825–832.        Roberts-Thomson et al., 1976. Gastroenterology. 71:57–61.        Rohrer et al., 1986. Antimicrob. Agents Chemother. 30:254–257.        Rolsma et al., 1998. J. Virol. 72:9079–9091.        Rueda et al., 1996. Biol. Chem. 377:599–601.        Rueda et al., 1998. J. Pediatr. 133:90–94.        Sorice et al., 1996. Parasite Immunol. 18:133–137.        Stevens et al., 1997. Exp. Parasitol. 86:133–143.        Suzuki, K. 1964. Life Sci. 3:1227–1233.        Underdown et al., 1981. J. Immunol.; 126:669–672.        Vazquez et al., 2001. BioFactors. 15:1–9.        Walterspiel et al., 1994. Pediatrics. 93:28–31.        Watarai et al., 1995. J. Vet. Med. Sci. 57:17–22.        Williams et al., 1980. J. Neurochem. 35:266–269.        Wolfe 1992. Clin. Microbiol. Rev. 5:93–100.        
Abbreviations used herein are as follows: GM1: II3 NeuAc-GgOse4Cer; GM2: II3 NeuAc-GgOse3Cer; GM3: II3 NeuAc-LacCer; GD1b: II3 (NeuAc)2-GgOse4Cer; GD3: II(NeuAc)2-LacCer; E. Coli: Escherichia coli; Gang-High: High concentration of ganglioside; Gang-Low: Low concentration of ganglioside; G: Giardia; LCPUFA: Long chain polyunsaturated fatty acids; NANA: N-Acetyl neuraminic acid; PBS: Phosphate buffered saline solution; SEM: Standard error of the mean; sIgA: Secretory immunoglobulin A; TG: Triglyceride; and TG+PUFA: Triglyceride containing polyunsaturated fatty acids.